Immune composition of erythrocyte-assisted hydrotalcite-based nano vaccine as well as preparation method and application of immune composition

Abstract

The invention relates to the technical field of medical vaccines, and discloses an immune composition of an erythrocyte-assisted hydrotalcite-based nano vaccine as well as a preparation method and application of the immune composition, the immune composition comprises aged erythrocytes and a hydrotalcite-based nano vaccine coated with a fresh erythrocyte membrane, and the hydrotalcite-based nano vaccine comprises a hydrotalcite-based adjuvant and an exogenous antigen. According to the preparation method and application of the immune composition of the hydrotalcite-based nano vaccine assisted by red blood cells in different states, efficient DC cell antigen cross presentation can be induced in the spleen, exogenous antigen specific immunostimulation is promoted, immune response of T cells is activated, solid tumor growth is directly inhibited, and the tumor volume inhibition rate reaches 60%.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical vaccines, and specifically relates to an immune composition of a red blood cell-assisted hydrotalcite-based nano vaccine and a preparation method and application thereof. BACKGROUND

[0002] In recent years, tumor immunotherapy has made breakthrough progress in the field of cancer treatment, but still faces many challenges: including low efficiency of tumor antigen delivery, insufficient immunogenicity, tumor microenvironment immunosuppression, and systemic toxic side effects. Existing nano vaccine systems mostly rely on synthetic carriers (such as liposomes, polymer nanoparticles) to deliver antigens and adjuvants, but these carriers often have problems such as poor targeting of immune cells, uncontrollable intracellular release, and low cross-presentation efficiency of antigens. In addition, a single delivery mode cannot break through the complex barriers of tumor immune escape, and it is urgent to develop a time-controllable multi-dimensional immune activation strategy.

[0003] Red blood cells, as natural biological carriers, have unique immune regulation characteristics. Studies have shown that senescent red blood cells expose phosphatidylserine (PS) and other "eat me" signals on their surface and reduce CD47 receptors and other "don't eat me" signals, which are then efficiently recognized and phagocytosed by antigen-presenting cells (APCs). The membrane surface of fresh red blood cells is rich in CD47 receptors, which releases "don't eat me" signals to avoid being phagocytosed by APC cells, thereby increasing the circulation time in the body. The characteristics of red blood cells in different states provide natural and diverse targeting pathways for antigen delivery, but traditional red blood cell drug loading technology has focused on drug packaging and has not fully explored the synergistic mechanism of different states of red blood cells and immune activation. For example, a Chinese patent with the patent number CN 114432261 A discloses a preparation method of a targeted vaccine, which is characterized by the following steps: S1: obtaining peripheral blood and storing it in an anticoagulant blood collection tube; S2: separating the red blood cells from the lymphocytes of the peripheral blood and collecting the red blood cells; S3: washing the collected red blood cells with 0.01M PBS or 0.9% physiological saline and centrifuging several times to obtain the red blood cells after the first centrifugation; S4: adding 1xPBS or 0.9% physiological saline with a concentration of 55.6%-83.3% to the centrifuged red blood cells for dilution, then adding the vaccine component, and storing at low temperature for 0.1-8 hours to obtain a mixture of red blood cells and vaccine; S5: adjusting the PBS concentration in the red blood cell and vaccine mixture to 0.01M and storing at low temperature for 0.1-8 hours; or adjusting the NaCl concentration in the red blood cell and vaccine mixture to 0.9% and storing at low temperature for 0.1-8 hours; S6: centrifuging to collect the red blood cells, then adding CD47 antibody or CpG and incubating for 0.1-8 hours; S7: washing the red blood cells with 0.01M PBS or 0.9% physiological saline and centrifuging several times to obtain the red blood cells after the second centrifugation, which are the red blood cell vaccine, i.e. the targeted vaccine. However, the problem of insufficient immune cell activation threshold has not been solved.

[0004] In addition, there are also research reports on the functional integration of different delivery systems, but lack of biological safety and clinical operability. For example, a Chinese patent with publication number CN 117379539 A discloses an immunocomposition of senescent red blood cell assisted hydrotalcite-based nano vaccine and its preparation method and application, which is characterized in that the immunocomposition comprises senescent red blood cells and a hydrotalcite-based nano vaccine, the hydrotalcite-based nano vaccine comprises a hydrotalcite-based adjuvant and an exogenous antigen; the hydrotalcite-based adjuvant is hexagonal flaky with a particle size of 150±30 nanometers; and the particle size of the hydrotalcite-based nano vaccine is 160±20 nanometers. The administration mode is intravenous injection, and the senescent red blood cells are injected intravenously first, and then the hydrotalcite-based nano vaccine is injected intravenously; and the preparation method of the senescent red blood cells is as follows: S1: after the fresh red blood cells are washed and diluted, the senescent red blood cells are obtained by in vitro heat stimulation. However, the senescent red blood cells induced by heat stimulation still partially retain membrane integrity, and the conformational change of CD47 may be more moderate. For example, although the “don't eat me” signal of CD47 is weakened, it can still partially bind to SIRPα on the surface of macrophages, delaying the phagocytosis speed, so that the targeting clearance time window of the immune system is longer, and the timing needs to be strictly controlled. Further, the safety concentration of hydrotalcite in the hydrotalcite-based adjuvant has always been a key factor limiting its use as an immunotherapy component, and moreover, its stability in vivo is also seriously challenged.

[0005] Aluminum adjuvants, including aluminum hydroxide or aluminum phosphate, can adsorb antigens, enhance the immune response of the body to antigens, and induce strong and long-lasting immune reactions, and have been widely used in the field of vaccines. Among them, layered double hydroxide (hydrotalcite, LDH) nanomaterials have attracted much attention in the field of vaccine delivery due to their high specific surface area, pH-responsive degradation, and inherent immune adjuvant effect. However, existing researches are mostly limited to the physical mixing of single LDH carriers and antigens, and have not achieved spatial positioning loading and timing release control of antigens / adjuvants.

[0006] The above research reports have not realized the optimized combination of red blood cells in different states for the application of vaccine delivery in immune activation. SUMMARY

[0007] The present application provides a red blood cell assisted hydrotalcite-based nano-vaccine immune composition and a preparation method and application thereof, which can significantly down-regulate the level of liver macrophages, so as to promote more hydrotalcite-based nano-vaccines to migrate to the spleen region rich in APCs; the APCs activated by the vaccine subsequently migrate to the B / T zone rich in B cells or T cells, activate immune cells and induce an anti-tumor immune response to attack tumors, thereby solving the problems of slow immune induction and insufficient immune response of a cancer treatment vaccine.

[0008] The technical scheme adopted by the present application is as follows: The present application provides a red blood cell assisted hydrotalcite-based nano-vaccine immune composition, which comprises senescent red blood cells and fresh red blood cell membrane coated hydrotalcite-based nano-vaccines.

[0009] Further, the particle size of the fresh red blood cell membrane coated hydrotalcite-based nano-vaccine is 70-250 nm.

[0010] Preferably, the hydrotalcite-based adjuvant is hexagonal flaky, with a particle size of 20-200 nm, and the particle size of the hydrotalcite-based nano-vaccine is 50-230 nm.

[0011] The hydrotalcite-based nano-vaccine prepared by the present application is easy to be taken up by cells, suitable for accumulation in the spleen, and exhibits obvious biological safety, and has no obvious toxicity to normal cells (DC2.4, RAW 264.7). In addition, in the immune composition provided by the present application, the senescent red blood cells are phagocytosed and removed by the macrophages of the spleen and liver, resulting in temporary saturation of the macrophages, thereby reducing their subsequent phagocytosis capacity. Furthermore, the fresh red blood cell membrane coated hydrotalcite-based nano-vaccine with different particle sizes further avoids phagocytosis by APC cells by virtue of the CD47 protein on the fresh red blood cell membrane, thereby significantly improving the in vivo circulation time. Moreover, the fresh red blood cell membrane coated hydrotalcite-based nano-vaccine is targeted to the spleen by virtue of the particle size (70-250 nm) and other physicochemical properties, promotes antigen presentation and cross-presentation, thereby enhancing the specific tumor killing function of T cells, and has great potential for improving tumor immunotherapy.

[0012] The present application provides a preparation method of a red blood cell assisted hydrotalcite-based nano-vaccine immune composition, comprising the following steps: 1) After the fresh red blood cells are washed and diluted with physiological saline, the senescent red blood cells are obtained by low temperature induction; 2) under inert gas protection, adding the mixed salt solution of aluminum chloride solution and magnesium chloride zinc solution into sodium hydroxide solution , stirring, and resuspending after centrifugation as a first mixed solution; 3) subjecting the first mixed solution obtained in step 2) to hydrothermal reaction, centrifuging, and taking the supernatant to obtain a hydrotalcite-based adjuvant; 4) mixing the hydrotalcite-based adjuvant obtained in step 3) with an exogenous antigen and ultrasonicating to obtain a hydrotalcite-based nano vaccine; 5) mixing the hydrotalcite-based nano vaccine obtained in step 4) with fresh red blood cell membranes and ultrasonicating to obtain fresh red blood cell membrane-coated hydrotalcite-based nano vaccines.

[0013] In the present application, the mixed salt solution of aluminum chloride and magnesium chloride is added into sodium hydroxide solution to generate a mixed solution of magnesium hydroxide and aluminum hydroxide. In the subsequent hydrothermal reaction, the aluminum / magnesium hydroxide slowly hydrolyzes to form a stable hydrotalcite-based adjuvant. Subsequently, the hydrotalcite-based adjuvant is mixed with an exogenous antigen and ultrasonicated to prepare a nano vaccine, which has the dual functions of dendritic cell activation and immunogenic cell death induction.

[0014] Further, the diluted red blood cells in step 1) are treated at 4℃ for 10-14 days in vitro.

[0015] Further, the preparation method of the mixed salt solution is as follows: 0.1-1 g of aluminum chloride is dispersed in 3.5-35 mL of an aluminum chloride solution in ultrapure water, and 0.1-1 g of magnesium chloride is dispersed in 3.5-35 mL of a zinc chloride solution in ultrapure water to obtain the mixed salt solution.

[0016] As preferred, the mixed salt solution is mixed and reacted with 0.1-1 g of sodium hydroxide solution dispersed in 10-100 mL under nitrogen protection, the stirring time is 5-60 min, and after centrifugation, 28-280 mL of ultrapure water is used for resuspension to obtain the first mixed solution.

[0017] Further, the temperature of the hydrothermal reaction in step 3) is 60-100℃, and the time is 6-24 h.

[0018] As preferred, the centrifugation speed in step 3) is 2000-6000 rpm, and the centrifugation time is 5-6 min.

[0019] As preferred, the exogenous antigen is ovalbumin (OVA) and To11-like receptor 9 agonist (CpG).

[0020] The application of a red blood cell-assisted hydrotalcite-based nano vaccine immune composition in the preparation of a drug for treating solid tumors.

[0021] Further, the solid tumor is one or several of melanoma, lymphoma, breast cancer, and bladder cancer.

[0022] Further, the administration mode of the immunological composition is intravenous programmed injection.

[0023] Further, the operation steps of the programmed injection are: 0.5-10 x 10 8 4-20 h later, intravenous injection of the fresh red blood cell membrane coated hydrotalcite-based nanovaccine.

[0024] The present application provides a preparation method and application of a hydrotalcite-based nanovaccine assisted by red blood cells in different states, aiming at the problems of slow immune induction and insufficient immune response in cancer therapeutic vaccines: first, pre-injection of aged red blood cells reduces the phagocytosis of liver macrophages to the subsequent injection of nanovaccine, and by adjusting the injection amount of aged red blood cells, the accumulation amount of nanovaccine in the spleen and tumor is increased; second, the preparation method of the fresh red blood cell membrane coated hydrotalcite-based nanovaccine is optimized, and by controlling the temperature and processing time of hydrothermal treatment, the size of the hydrotalcite-based adjuvant is controlled, so that it is more suitable for accumulation in the spleen, and by means of the immune cell activation function of the nanovaccine, rapid and strong immune stimulation is induced.

[0025] In summary, due to the adoption of the above technical solutions, the present application has the following advantages: 1) The present application uses aged red blood cells to pretreat mice, and the subsequent injection of hydrotalcite-based nanovaccine can simultaneously and efficiently accumulate in the spleen, and the fresh red blood cell membrane coated hydrotalcite-based nanovaccine can realize rapid endosome escape, effectively release antigens to the cytoplasm of DC cells, and promote antigen presentation and cross-presentation.

[0026] 2) The present application uses a preparation method and application of a hydrotalcite-based nanovaccine assisted by red blood cells in different states, which can induce efficient DC cell antigen cross-presentation in the spleen, promote exogenous antigen specific immune stimulation, activate T cell immune response, and directly inhibit solid tumor growth, with a tumor volume inhibition rate of 60%.

[0027] 3) The hydrotalcite nanovaccine in the present application has a simple synthesis process, clear composition and structure, can realize good cell uptake and spleen targeting effect, and has high purity.

[0028] 4) The hydrotalcite-based nanovaccine obtained by the present application has no obvious toxicity to normal cells and good biological safety. DETAILED DESCRIPTION

[0029] The present application will be described by way of example and with reference to the accompanying drawings, in which: Figure 1 TEM image of the hydrotalcite-based adjuvant prepared for Example 1.

[0030] Figure 2 TEM image of hydrotalcite-based nanovaccine prepared for Example 1.

[0031] Figure 3 TEM image of fresh red blood cell membrane-coated hydrotalcite-based nanovaccine prepared for Example 1.

[0032] Figure 4 Antigen loading capacity evaluation image of fresh red blood cell membrane-coated hydrotalcite-based nanovaccine in Example 1.

[0033] Figure 5 Cytotoxicity evaluation image of fresh red blood cell membrane-coated hydrotalcite-based nanovaccine in Example 1.

[0034] Figure 6 Antigen presentation level evaluation image of fresh red blood cell membrane-coated hydrotalcite-based nanovaccine in Example 1.

[0035] Figure 7 Effect image of fresh red blood cell membrane-coated hydrotalcite-based nanovaccine in Example 1 promoting T cell proliferation.

[0036] Figure 8 Spleen accumulation amount change image of hydrotalcite-based nanovaccine after aging red blood cell pretreatment in Example 6.

[0037] Figure 9 In vivo tumor inhibition effect evaluation image of hydrotalcite-based nanovaccine after aging red blood cell pretreatment in Example 7.

[0038] Figure 10 Spleen DC antigen presentation effect evaluation image of hydrotalcite-based nanovaccine after aging red blood cell pretreatment in Example 7.

[0039] Figure 11 Exogenous antigen-specific immune effect evaluation image of hydrotalcite-based nanovaccine after aging red blood cell pretreatment in Example 7. DETAILED DESCRIPTION

[0040] All features disclosed in this specification, and / or all steps of any methods or processes disclosed in this specification, can be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0041] The present application will now be described in detail below with reference to the examples.

[0042] Example 1: Synthesis and characterization of fresh red blood cell membrane-coated hydrotalcite-based nanovaccine (1) 0.63 g magnesium chloride was dispersed in 5 mL ultrapure water to obtain a magnesium chloride aqueous solution; 0.24 g aluminum chloride was dispersed in 5 mL ultrapure water to obtain an aluminum chloride aqueous solution; then the mixed salt solution was obtained by mixing; 0.72 g sodium hydroxide was dispersed in 20 mL ultrapure water; the mixed salt solution and the sodium hydroxide solution were mixed and reacted for 30 min under argon protection, and then washed twice with ultrapure water and resuspended with 40 mL ultrapure water by centrifugation.

[0043] (2) The mixture in step (1) was transferred to a 50 mL volume high-pressure hydrothermal kettle, heated to 100°C, and reacted for 6 h. After the hydrothermal kettle was taken out and cooled to room temperature, 4000 g was centrifuged for 5 min, and the supernatant was taken to obtain a hydrotalcite-based adjuvant (LDH).

[0044] (3) The hydrotalcite-based adjuvant was mixed with ovalbumin and adsorbed by ultrasonic to form a hydrotalcite-based nano vaccine (LO).

[0045] (4) The hydrotalcite-based nano vaccine was mixed with fresh red blood cell membranes at a mass ratio of 50:1, and adsorbed by ultrasonic to form a fresh red blood cell membrane-coated hydrotalcite-based nano vaccine (RLO).

[0046] The TEM characterization of water LDH, LO and RLO is shown in Figure 1 , 2 and 3, respectively. The particle size of the synthesized hydrotalcite-based adjuvant is about 100±20 nm, the size of the hydrotalcite-based nano vaccine is about 120±20 nm, and the fresh red blood cell membrane-coated hydrotalcite-based nano vaccine is about 140±20 nm.

[0047] Example 2: Evaluation of the antigen loading capacity of the fresh red blood cell membrane-coated hydrotalcite-based nano vaccine The hydrotalcite nano-aluminum adjuvant was co-incubated with different concentrations of OVA antigen protein solution for 15 min, and the supernatant was taken by centrifugation at 15000 rpm to determine the concentration of free protein. The adsorption capacity of the above RLO to antigen protein was analyzed by fitting the Langmuir isothermal adsorption model Qe = QmCe / (1 / K+Ce).

[0048] The results are shown in Figure 4 From Figure 4 , it can be seen that the maximum adsorption amount of antigen protein per milligram of RLO is 0.17 mg of OVA protein, which proves that the above nucleotide-loaded hydrotalcite nano-aluminum adjuvant can efficiently adsorb antigen protein and is beneficial to the delivery of antigen protein to the intracellular environment to promote antigen presentation.

[0049] Example 3: Toxicity of hydrotalcite-based nano vaccine to dendritic cells and macrophages (1) DC2.4 and RAW264.7 cells were seeded in a 96-well plate at a density of 1×104 cells / well and cultured overnight.

[0050] (2) Then the medium was replaced with fresh medium containing RLO prepared in Example 1, and the cell viability was detected by MTT method after 24 h of culture.

[0051] (3) The average value of OD value of 6 holes of each sample was calculated, and the cell viability (%) was calculated according to the following formula: Cell viability (%) = average value of OD value of sample group / average value of OD value of blank control group x 100%.

[0052] The results are shown in Table 1. Figure 5 The RLO prepared in Example 1 has low toxicity to normal cells DC2.4 and RAW 264.7 cells.

[0053] Example 4: Enhancement of antigen presentation of dendritic cells by fresh red blood cell membrane coated hydrotalcite-based nanovaccine DC2.4 cells were inoculated in a 24-well plate overnight, and then treated with OVA, LDH, LO and RLO (200 μg mL-1) prepared in Example 1 for 24 h. The level of antigen presentation was detected by using a fluorescently labeled exogenous antigen-specific antibody.

[0054] The results are shown in Table 2. Figure 6 After treatment with RLO prepared in Example 1, the level of antigen presentation of DC cells was enhanced.

[0055] Example 5: Effect of fresh red blood cell membrane coated hydrotalcite-based nanovaccine on promoting T cell proliferation DC cells treated with hydrotalcite-based nanovaccine in Example 4 were co-incubated with OT-I T cells for 72 hours, and the green fluorescence attenuation of CFSE-labeled OT-I T cells was detected by flow cytometry to characterize the proliferation of T cells.

[0056] The results are shown in Table 3. Figure 7 The hydrotalcite-based nanovaccine significantly increased the proliferation ratio of OT-I T cells by significantly enhancing the antigen presentation of DC. This indicates that the hydrotalcite-based nanovaccine delivery of OVA antigen activated DC cells can enhance cross-antigen presentation and promote the proliferation of cytotoxic T cells.

[0057] Example 6: Enhancement of accumulation of spleen and tumor vaccine by senescent red blood cell pretreatment (1) 6 x 108 senescent red blood cells were intravenously injected into mice for 24 h to saturate the macrophages in the liver of the mice.

[0058] (2) After 24 h, fresh red blood cell membrane hydrotalcite-based nanovaccine was intravenously injected to increase the accumulation of nanovaccine in the spleen.

[0059] The accumulation of the hydrotalcite-based nanovaccine in the spleen and tumor was detected, and the detection results are shown in Figure 8 Fresh red blood cell membrane coating can increase the accumulation of the hydrotalcite-based nanovaccine in the spleen. After further pretreatment of the aged red blood cells, the accumulation of the vaccine in the spleen is significantly increased.

[0060] Example 7: Anti-tumor effect of the hydrotalcite-based nanovaccine immune composition assisted by aged red blood cells (1) Evaluation of in vivo tumor inhibition effect Twenty-four C57 mice were randomly divided into four groups, six in each group. They were saline, OVA, fresh red blood cell membrane coated hydrotalcite-based nanovaccine (RLO), and aged red blood cells + fresh red blood cell membrane coated hydrotalcite-based nanovaccine treatment (sRBC+RLO). The tumor volume (V) was measured every two days, and the calculation formula was: V = L x W2 / 2, W and L were the longest and widest diameters of the tumor, respectively. The tumor volume change was monitored by comparing the tumor volume with the original volume.

[0061] The results are shown in Figure 9 The inhibition rate of the aged red blood cells + fresh red blood cell membrane coated hydrotalcite-based nanovaccine treatment on the in situ tumor was 73%, which proved that the implementation method of the aged red blood cells + fresh red blood cell membrane coated hydrotalcite-based nanovaccine had good tumor inhibition effect.

[0062] (2) Evaluation of antigen presentation level at animal level After the mice were sacrificed after 2 immunizations, the spleen was removed to obtain a spleen cell suspension. The level of antigen presentation was detected by using a fluorescently labeled exogenous antigen specific antibody.

[0063] The results are shown in Figure 10 The aged red blood cells + fresh red blood cell membrane coated hydrotalcite-based nanovaccine treatment group showed the highest antigen presentation level.

[0064] (3) Evaluation of exogenous antigen specific immune response level After the mice were sacrificed after 2 immunizations, the spleen was removed to obtain a spleen cell suspension. In vitro, OVA (exogenous antigen) was used to stimulate memory cells in the spleen, and the exogenous antigen specific immune response level was detected by flow cytometry analysis.

[0065] The results are shown in Figure 11 The aged red blood cells + hydrotalcite-based nanovaccine treatment group induced the exogenous antigen specific immune response, and the effect was the best.

[0066] The above embodiments only express the specific implementation of the present application, which is described in more detail and specifically, but cannot be understood as a limitation to the protection scope of the present application. It should be noted that for those skilled in the art, without departing from the technical concept of the present application, a number of modifications and improvements can be made, which are all within the protection scope of the present application.

Claims

1. An immunogenic composition for a erythrocyte-assisted hydrotalcite-based nanovaccine, characterized in that, The immune composition comprises senescent red blood cells and fresh red blood cell membrane-coated hydrotalcite-based nanovaccines, wherein the hydrotalcite-based nanovaccines comprise hydrotalcite-based adjuvants and exogenous antigens.

2. The immunizing composition for a erythrocyte-assisted hydrotalcite-based nanovaccine according to claim 1, characterized in that, The fresh red blood cell membrane-coated hydrotalcite-based nanovaccine has a particle size of 70-250 nm.

3. A method for preparing an immunogenic composition of a erythrocyte-assisted hydrotalcite-based nanovaccine according to any one of claims 1 to 2, characterized in that, Includes the following steps: 1) Fresh red blood cells were washed and diluted with physiological saline, and then induced to become senescent red blood cells at low temperature; 2) Under inert gas protection, add a mixed salt solution of aluminum chloride solution and magnesium zinc chloride solution to sodium hydroxide solution. Stir, centrifuge, and resuspend as the first mixed solution; 3) The first mixed solution obtained in step 2) is subjected to hydrothermal reaction, centrifuged, and the supernatant is taken to obtain the hydrotalcite-based adjuvant; 4) The hydrotalcite-based adjuvant obtained in step 3) is mixed with the exogenous antigen and sonicated to obtain a hydrotalcite-based nanovaccine; 5) Mix the hydrotalcite-based nanovaccine obtained in step 4) with fresh red blood cell membrane and sonicate to obtain a hydrotalcite-based nanovaccine coated with fresh red blood cell membrane.

4. The method for preparing an immunogenic composition for a erythrocyte-assisted hydrotalcite-based nanovaccine according to claim 3, characterized in that, The diluted red blood cells from step 1) were subjected to in vitro low-temperature treatment at 4°C for 10-14 days.

5. The method for preparing an immunogenic composition for a erythrocyte-assisted hydrotalcite-based nanovaccine according to claim 3, characterized in that, The mixed salt solution is prepared by mixing 0.1-1g of aluminum chloride dispersed in 3.5-35mL of ultrapure water in an aluminum chloride solution and 0.1-1g of magnesium chloride dispersed in 3.5-35mL of ultrapure water in a zinc chloride solution to obtain a mixed salt solution.

6. The method for preparing an immunogenic composition for a erythrocyte-assisted hydrotalcite-based nanovaccine according to claim 5, characterized in that, The hydrothermal reaction temperature is 60–100℃, and the time is 6–24 hours.

7. The use of an immunoassay composition of a erythrocyte-assisted hydrotalcite-based nanovaccine according to any one of claims 1 to 2 in the preparation of a medicament for treating solid tumors.

8. The application of the immunizing composition for a erythrocyte-assisted hydrotalcite-based nanovaccine according to claim 7, characterized in that, The solid tumor is one or more of the following: melanoma, lymphoma, breast cancer, and bladder cancer.

9. The application of the immunizing composition for a erythrocyte-assisted hydrotalcite-based nanovaccine according to claim 7, characterized in that, The immune composition is administered via programmed injection.

10. The application of the immunizing composition for a erythrocyte-assisted hydrotalcite-based nanovaccine according to claim 9, characterized in that, The procedure for the programmed injection is as follows: Pre-inject 0.5–10 × 10⁻⁶ intravenously. 8 Four to 20 hours later, senescent red blood cells were intravenously injected with a hydrotalcite-based nanovaccine coated with fresh red blood cell membranes.

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